Dengue virus (DV), a positive stranded RNA virus, is transmitted by the mosquitoes, and is the cause of a growing public health problem. Approximately 60-80 million persons are infected annually, and rates of infection are as high as 6% in some areas. The virus can be divided into 4 serotypes (DEN 1-4), and all 4 serotypes circulate in Caribbean, Asia and the Americas. Infection with one strain does not provide protective immunity against other strains. There are several reasons for the lack of an effective vaccine against DV. It is known that pre-existing non neutralizing antibodies indeed enhance the virus replication during secondary infection. In view of this an ideal vaccine will include only protective epitopes so that deleterious effect can be avoided. However idea of construction of a polyepitope vaccine cannot be realized at this time because adequate numbers of immune targets have not been identified in all 4 serotypes. This application deals with identification of such targets but our proposal remains limited to T cell epitopes. We plan to identify CTL and/or T-helper epitopes in prM-E and NS-1 protein of the DEN-3. Our central hypothesis is that prM-E and NS-1 protein are targets of T cell mediated immune response during natural infection with DEN-3, and there are T-helper and CTL epitopes in these proteins. We will develop DV3-specific T cell lines and clones. These clones will be characterized for CD4 and CD8 phenotype and used for the identification of T-helper and CTL epitopes in ELISPOT assays. The clones will be first tested against overlapping peptides encompassing full length prM-E and NS1, followed by screening against deletion peptides. The HLA restriction element for the newly identified epitopes will be determined. We will also determine functional properties of the CD4 and CD8 T cell clones like cytokine secretion pattern, ability to block/reduce replication of DEN-3 as well as other serotypes.